The goal of Project 2 is to produce real-time multiplex PCR for enteropathogen diagnostic tests for public health laboratories. We will focus on the highest priority protozoan and enteric Category B agents as defined by the recent Blue Ribbon Panel, namely Cryptosporidia, Entamoeba, Giardia and Shigella. Preliminary studies have pioneered methodology for DNA extraction from stool and PCR-based stool tests for the identification and DNA fingerprinting of Entamoeba. We will build on our preliminary studies to develop real-time multiplex PCR using molecular beacons, FRET and/or Taqman probes. The goal will be to have the capability working with a single stool DNA sample to identify in a multiplex format the 3 parasites and their medically-significant genotypes and then add Shigella detection to the format so that all 4 of the major bacterial/parasitic Category B agents are detectable.